Bibliographic details of the publications referred to by the author in this specification are collected at the end of the description.
Reference to any prior art, in this specification is not, and should not be taken as an acknowledgment or any form of suggestion that this prior art is common general knowledge or forms part of the common general knowledge in Australia or any other country.
Molecular biology and genomic research is rapidly changing the scope as well as the focus of genetic research into causes of inherited diseases. With the rapid increase in primary DNA sequence information, the direct comparison of test DNA to known sequence as a way to characterise mutations is a methodology in need of development.
One approach to this problem is to sequence the unknown target DNA. DNA sequencing technology has become more commonplace and less cumbersome in recent years, but this approach is not suitable for high throughput mutation detection scanning due to low speed and high cost.
A second approach has exploited sequence specific conformational differences in either native (Orita, et al., 1989) or denaturing gradient (Foode. R, 1994) polyacrylamide gel electrophoresis (PAGE). These methods, in general, are relatively easy to perform, but are slow, and have a high rate of false negatives.
An elegant, but insensitive, approach to DNA mutation detection targets not DNA, but rather the DNA's expressed product. Functional assays of p53 (Schwartz, H. et al., 1998) proteins have been developed in which in vivo testing of cloned DNA in yeast can efficiently identify those chimeras carrying mutated gene fragments, as long as the product is nonfunctional. A similar in vitro approach, the protein truncation test (Roest, P. A. et al., 1993) has been developed for several cancer genes. This test, as the name implies, uses mRNA as a substrate in a transcription/translation experiment in which the synthesized protein product is sized by PAGE. The presence of a peptide shorter than a wild type control is evidence of a mutation resulting from either a deletion or a stop codon. These methods are quite useful for characterization of this type of major DNA aberration, but single base missense mutations are seldom detectable.
The fourth major category of mutation detection methodology depends on nucleic acid hybridization between known and unknown paralogs. If two or more different nucleic acid species are present in this reaction, heteroduplexes can form in which one or more mismatched base pairs exist. These mismatches can then be detected by enzymatic (Youil, R. et al., 1995; Parsons, B. L., 1997; Lu, A. L., 1992; Myers, R. M. et al., 1985) or chemical (Cotton R. G. et al., 1988) methods. While some of these methods are potentially quite useful for high-throughput screening because the detection of mismatches can be performed without gel electrophoresis (Parsons, B. L., 1997; Lu, A. L., 1992), the methods are not easily multiplexed. This approach, while simple in theory, has also spawned several new technologies for the detection of base mutations including DNA chips (Sosnowski, R. G. et al., 1997; Hacia, J. G. et al., 1996), mismatched duplexes found by denaturing high performance liquid chromatography (O'Donovan M. C. et al., 1998), and in-tube kinetic assays (Holland P. M. et al., 1991; Ririe, K. M. et al., 1997).
In the work leading up to the present invention, the inventors developed a DNA mutation detection system which applies competitive hybridization and differential labelling to produce a method capable of discriminating between single base mismatches in oligonucleotides. The method of the present invention is also capable of being multiplexed and automated.